Contribution of glutamate dehydrogenase to mitochondrial glutamate metabolism studied by C-13 and P-31 nuclear magnetic resonance

The relative contribution of glutamate dehydrogenase (GDH) and the aminotra nsferase activity to mitochondrial glutamate metabolism was investigated in dilute suspensions of purified mitochondria from potato (Solanum tuberosum ) tubers. Measurements of glutamate-dependent oxygen consumption by mitocho ndria in different metabolic states were complemented by novel in situ NMR assays of specific enzymes that metabolize glutamate, First, a new assay fo r aminotransferase activity, based on the exchange of deuterium between deu terated water and glutamate, provided a method for establishing the effecti veness of the aminotransferase inhibitor amino-oxyacetate in situ, and thus allowed the contribution of the aminotransferase activity to glutamate oxi dation to be assessed unambiguously, Secondly, the activity of GDH in the m itochondria was monitored in a coupled assay in which glutamine synthetase was used to trap the ammonium released by the oxidative deamination of glut amate, Thirdly, the reversibility of the GDH reaction was investigated by m onitoring the isotopic exchange between glutamate and [N-15]ammonium. These novel approaches show that the oxidative deamination of glutamate can make a significant contribution to mitochondrial glutamate metabolism and that GDH can support the aminotransferases in funnelling carbon from glutamate i nto the TCA cycle.